-
Planta (1994)195:63-69 P l ~ I l t ~
�9 Springer-Verlag 1994
Polarity induction versus phototropism in maize: Auxin cannot
replace blue light Peter Nick, Eberhard Schfifer
Institut f/Jr Biologic II, Sch/inzlestrasse 1, D-79104 Freiburg,
Germany
Received: 2 March 1994 / Accepted: 18 April 1994
Abstract. In a previous study (Nick and Sch~fer 1991, Planta
185, 415-424), unilateral blue light had been shown, in maize
coleoptiles, to induce phototropism and a stable transverse
polarity, which became detectable as stable curvature if
counteracting gravitropic stimulation was removed by rotation on a
horizontal clinostat. This response was accompanied by a
reorientation of cortical microtubules in the outer epidermis (Nick
et al. 1990, Planta 181, 162-168). In the present study, this
stable transverse polarity is shown to be correlated with stabili-
ty of microtubule orientation against blue light and changes of
auxin content. The role of auxin in this stabil- isation was
assessed. Although auxin can induce reorien- tation of microtubules
it fails to induce the stabilisation of microtubule orientation
induced by blue light. This was even true for gradients of auxin
able to induce a bending response similar to that ellicited by
phototropic stimulation. Experiments involving partial irradiation
demonstrated different perception sites for phototropism and
polarity induction. Phototropism starts from the very coleoptile
tip and involves transmission of a signal (auxin) towards the
subapical elongation zone. In con- trast, polarity induction
requires local action of blue light in the elongation zone itself.
This blue-light response is independent of auxin.
Key words: Auxin - Blue light - Coleoptile - Microtubule -
Phototropism - Transverse polarity - Zea
Introduction
Plants can adapt their development to changing environ- mental
conditions. This implies the ability to sense and process
environmental signals and the ability to tune cel- lular
morphogenesis with this processed information
Correspondence to: P. Nick; FAX: 49 (761)203 4217
(Mohr 1972). Microtubules are candidates for the link between
signal transduction and morphogenesis: they re- orient swiftly in
response to hormones, light, gravity and endogenous factors (Iwata
and Hogetsu 1989; Nick et al. 1990b, 199 lb; Sakiyama and Shibaoka
1990; Zandomeni and Schopfer 1993). They appear to guide the
directional deposition of cellulose microfibrils, an important
mecha- nism of growth control (Robinson and Quader 1982).
Phototropism of coleoptiles is one of the most sensi- tive and
rapid morphogenetic responses known, with a lag of only 20-30 min
(Iino 1988). In maize coleoptiles, reorientation of cortical
microtubules in the illuminated coleoptile flank precedes the
bending response to pho- totropic stimulation (Nick et al. 1990b).
An early model postulated that the light-induced depletion of auxin
in the illuminated coleoptile flank should trigger the ob- served
microtubule orientation from transverse to longi- tudinal.
Deposition of cellulose microfibrils in the longi- tudinal
direction should then produce a stiff cell wall in the illuminated
flank and, in consequence, an inhibition of growth. However, a more
detailed analysis demon- strated that conspicuous curvatures could
arise without or even against gradients of microtubule orientation,
and that gradients of microtubule orientation can be induced that
are not followed by bending (Nick et al. 1991a). Thus, microtubule
reorientation was found to be neither necessary nor sufficient for
tropistic bending. The re- sponses are only correlated, not
causally linked.
In addition to the tropistic response, blue light can evoke a
stable transverse polarity in the direction of stim- ulation (Nick
and Sch/ifer 1988, 1991). This polarity can withstand opposing
gravitropic or phot0tropic stimuli for many hours and becomes
manifest as a stable, long- lasting curvature, if the gravitropic
counterstimulation experienced by curved plants is removed by
rotation on a horizontal clinostat. This transverse polarity
evolves from a labile precursor, which becomes stable 90 min af-
ter induction (Nick and Sch/ifer 1991). Thus, unilateral
irradiation by a pulse of blue light triggers a triple re- sponse:
phototropic curvature (lag 20-30 min, Iino 1988),
-
64 P. Nick and E. Schfifer: Polarity induction versus
phototropism
induc t i on of a s table t ransverse po l a r i t y (lag 90
min, N ick and Schfifer 1991), and r eo r i en t a t i on of cor t
ica l micro- tubules (lag 10-20 min, N ick et al. 1990b). It has
been shown prev ious ly (Nick et al. 1991a) tha t m ic ro tubu l e
o r i en t a t i on and p h o t o t r o p i c cu rva tu re are not
a lways cor re la ted . Thus, the p resen t pub l i ca t i on a t t
emp t s to clarify the connec t ion be tween m i c r o t u b u l e
o r i en ta t ion and s table t ransverse polar i ty .
Materials and methods
Plants and light conditions. Seedlings of maize (Zea mays L. cv.
Brio42HT; Asgrow, Bruchsal, Germany) were grown under 0.4 W.m 2 red
light for 2 d at 25~ and then kept in darkness for one further day.
This treatment yielded plants with straight coleop- tiles, since
mesocotyl elongation and nutations are suppressed by red light
(Kunzelmann and Schfifer 1985). All experiments were performed in a
symmetrical and saturating red background light (2.5 W.m 2) to
level out possible effects of phytochrome gradients induced by
phototropic stimulation (Hofmann and Schfifer 1987). For details on
growth conditions and selection procedures refer to Nick and
Schfifer (1988) and Nick et al. (1992).
Stimulation treatments. The protocol for alternating stimulation
in- volved two unilateral light pulses of identical fluence (1.9
~tmol.m 2, 30 s) but opposing direction, parallel to the shorter
diameter of the coleoptile. The second, counteracting, pulse was
administered either 1 or 2 h after the first, inducing, light
pulse. In one set of experi- ments, the first pulse was applied not
unilaterally, but from above by means of a mirror. Except for
during the irradiation treatments, the plants were kept rotating on
a horizontal clinostat at 0.5 rpm until response evaluation as
described in detail in Nick and Schfifer (1991). In a variation of
this procedure the counteracting light pulse was replaced by
decapitation and subsequent incubation in water or a solution of
0.1 mM indole-3-acetic acid (IAA) according to Nick et al. (1992).
Energy fluxes were determined as described previ- ously (Nick and
Schfifer 1988).
Localized illumination. The procedure described above was varied
by giving the first light pulse only to specific regions of the
intact coleoptile (see Fig. 3). This was achieved by using a
light-piping device (Flexilux 150 HL; Sch611y Fiberoptik,
Denzlingen, Ger- many). The inducing light pulse was given either
to the very tip (treatment 2 in Fig. 3) or 20 mm below the tip
(treatment 3 in Fig. 3). The stability of the resulting curvature
was then tested by a counter- directed light pulse at variable time
intervals after induction. This counterpulse was applied to the
entire length of the coleoptile. The fluence of the inducing light
spot was reduced to 0.8 gmol.m 2 blue light with a spot diameter of
0.5 mm to minimize light-piping effects within the tissue (Mandoli
and Briggs 1982). The counterpulse was equal in fluence to the
inducing pulse. A control experiment record- ed the response to
0.85 gmol.m 2 blue light, where both, inducing and opposing light
pulses were given over the entire length of the coleoptile (see
Fig. 3, treatment 1). Both light pulses were of maxi- mally 30 s
duration. A second control assayed the tropistic respons- es to tip
and base illumination for omission of the counterstimula- tion.
Each datum point in Fig. 3 represents the average of 12 indi-
vidual seedlings.
Response evaluation. Phototropic curvature was determined using
a simple xerographic method (Nick and Schfifer 1988). Cortical mi-
crotubules were stained by means of immunofluorescence. Coleop-
tile segments (length 20 mm, 2 mm below the tip) were excised, the
primary leaf was discarded, and the side facing the inducing pulse
was marked by an incision. After prefixation for 45 min at room
temperature in 3.2% (w/v) paraformaldehyde in microtubule-stabi-
lizing buffer (0.1 M 1,4 piperazine-diethanesulfonic acid, 1 mM
MgCI 2, 5 mM ethylene
glycol-bis-([3-aminomethyl-ether)-N,N,N',N'-
tetraacetic acid, 0.2 % Triton X 100, pH 6.8), tangential
sections were cut under a drop of microtubule-stabilizing buffer
from the fiat sides of the coleoptile and collected separately with
respect to coleoptile flank. Then fixation in the same solution was
continued for a further 40min. After three washings in the same
buffer without paraformaldehyde the sections were incubated for 20
min at room temperature with goat normal serum (Nordic Immunology,
Tilburg, The Netherlands; diluted 1:20 in phosphate-buffered sa-
line, PBS) and then treated for 1 h at 37~ with a mouse monoclon-
al antibody raised against [3-tubutin (Amersham, UK) diluted 1 :
1000 in PBS. The sections were washed with PBS and incubated for 50
min at 37~ with a fluorescein-isothiocyanate-labeled sec- ondary
antibody (anti-mouse immunoglobulin G from sheep, 1:20 diluted in
PBS, Amersham), washed again and then mounted in an antifading
agent (Citifluor, Amersham, UK) with the outer face of the
epidermis facing upwards. They were viewed under a fluores- cence
microscope (Orthopan, Leitz, Wetzlar, Germany) and pho- tographed
on Kodak TriX Pan 400 ASA film (Kodak, Rochester, New York, USA).
Since there seems to be no preferential handed- ness to the
obliqueness of microtubules within the tissue (Nick et al. 1990b),
their orientation was scored according to four classes with 0 ~
designating transverse microtubules, 30 ~ slightly oblique micro-
tubules, 60 ~ steeply oblique microtubules and 90 ~ longitudinal
mi- crotubules. Frequency distributions were constructed from the
data from 20-45 plants corresponding to at least two independent
sets of experiments and mean orientation calculated from these
distribu- tions (Tables 1-5).
Results
Orientation o f microtubules is stable 2 h after irradiation
with blue light. Fol lowing sequent ia l s t imula t ion with two o
p p o s i n g blue- l ight pulses of equal s t rength (1.9 ~tmol- m
2, 30 s), cu rva tu re deve loped under cond i t ions of sym- metr
ic gravi ty dur ing ro t a t i on on an ho r i zon ta l c l inos ta
t (Fig. 1). W h e n the o p p o s i n g pulse was given 60 min
after the induc ing st imulus, final cu rva tu re was d o m i n a t
e d by this o p p o s i n g s t imula t ion (Fig. 1, left panel).
Mic ro - tubules, which had been t ransverse on bo th f lanks of
the coleopt i le at the t ime of induct ion , were found to be
long- i tud ina l in the i l lumina ted side and t ransverse in the
shaded side 60 min la ter (Fig. 1, left panel , Fig. 2). This g rad
ien t of mic ro tubu le o r i en ta t ion was reversed briefly
after the app l i ca t ion of the counterpulse , fol lowing the
invers ion of curvature . Eventual ly , mic ro tubu les became long
i tud ina l in the shaded side (facing the second pulse), where
they had been t ransverse, whereas in the f lank fac- ing the first
s t imulus, they tu rned back f rom long i tud ina l to t ransverse
(Fig. 1, left panel , Fig. 2). Thus, ne i ther the p h o t o t r o
p i c response no r the g rad ien t of m ic ro tubu l e o r i en ta
t ion induced by the first pulse showed any stabil i - ty aga ins t
an oppos ing s t imula t ion . In fact, a c o m p a r i s o n with
a control , where the first pulse had been omi t ted , gives the
impress ion tha t the coun te rpu l se had erased all t races of
the or iginal s t imula t ion .
A fundamen ta l difference was observed when the coun te rpu l
se was admin i s t e red 90 min after the induc ing s t imulus
(Fig. 1, r ight panel). A l t h o u g h the second pulse cou ld con
t ro l bend ing f o r the first hou r after counte r - s t imula t
ion , bend ing eventua l ly was reversed and p lan ts curved t ow a
rds the first l ight pulse. The final resul t was ind i s t ingu
ishab le f rom con t ro l exper iments in which the coun te rpu l
se had been omi t ted . The grad ien t of micro-
-
P. Nick and E. Sch/ifer: Polarity induction versus phototropism
65
Time (min)
~ ~ ~ 1 2 0 ~ J ~
~ ~ ~ 1 8 0 ~ J ~
8 0 ~ ~
Frequency (%)
Fig. 1. Stabilisation of transverse polarity and microtubule
orienta- tion by unilateral blue light in Zea coleoptiles.
Left-hand panel: Labile polarity for counterstimulation 60 min
after phototropic in- duction. Right-hand panel: Stable polarity
for counterstimulation 90 min after phototropic induction. The
shadowgraphs show the bending response of a typical seedling.
Frequency distributions rep- resent the orientation of cortical
microtubules in the epidermis in the corresponding coleoptile
flank, respectively with 0 ~ transverse, and 90 ~ longitudinal
microtubules
tubule orientation induced by the first light pulse did not
reveal any effects of the counterstimulus. Microtubules remained
longitudinal in the side facing the inducing stimulus and
transverse in the opposite coleoptile flank throughout the
experiment, i.e. even during the short pe- riod when the seedlings
transiently bent towards the counterpulse (Fig. 1, right panel). It
thus appeared that, 90 min after induction, both the transverse
polarity in the direction of the light (Nick and Schfifer 1988,
1991) and the gradient of microtubule orientation had attained sta-
bility against the opposing stimulation.
This apparent stability of microtubule arrays against
counterstimulation could be caused by a loss of respon- siveness to
blue light 90 min after irradiation. Alterna- tively, microtubule
orientation itself might become stable at this time. With the
intention of deciding between these possibilities, the second light
pulse was replaced by a different treatment: changes in the content
of endoge- nous auxin or exogenous IAA, respectively. Although 1 h
after a unilateral light pulse a clear gradient of micro- tubule
orientation could be detected, this gradient (longi- tudinal
microtubules in the illuminated side, transverse microtubules in
the shaded side) could be eliminated by changing the content of
auxin/IAA (Table 1). Depletion of endogenous auxin for 1 h yielded
longitudinal micro- tubules in both sides, incubation with
saturating concen- trations of IAA yielded transverse microtubules
in both sides. Identical results were obtained for unstimulated
coleoptiles (Table 1). Two hours after induction, the light-
induced gradient of microtubule orientation had become stable this
time against changes in the content of auxin/ IAA (Table 1). It
should be emphasized that this stabili- sation of a gradient
extended to microtubule orientation in the shaded side. Those
microtubules maintained their transverse orientation against
depletion of endogenous auxin, although they had not experienced a
light-induced reorientation response. This means that a loss of
respon- siveness to blue light can be ruled out as an explanation
and that microtubule orientation per se has become sta- ble.
Fig. 2. Inversion of the gradient in the orientation of cortical
mi- crotubules of Zea coleoptiles by phototropic counterstimulation
applied 60 min after induction. Cortical microtubules stained by
immunofluorescence in the lit (L) and shaded (S) flanks of a
coleoptile 60 min after pho- totropic stimulation (left). A
counteracting phototropic stim- ulation of equal strength was
administered at this time on the shaded side of the coleoptile. The
gradient in the orientation of microtubules between lit and shaded
sides had been reversed by 120 min after the original light pulse
(right), corresponding to the left column in Fig. 1. Bar = 10 gm;
•
-
66 P. Nick and E. Schiller: Polarity induction versus
phototropism
Table 1. Stabilisation of microtubule orientation against auxin
or IAA after phototropic induction. Zea coleoptile segments were
ex- cised at variable time intervals after phototropic induction
and incubated for 1 h either in water (causing depletion of
exogenous auxin) or in 10 laM IAA. The gradient in the orientation
of cortical
microtubules became stable 2 h after induction. Values represent
mean _+ SE of frequency distributions constructed for microtubule
orientation, with 90 ~ indicating longitudinal orientation and 0 ~
transverse orientation
Time after induction (At)
Control: intact coleoptiles
Tropistic curvature (o)
Microtubule orientation (~
Lit side Shaded side
Excision at At and incubation in water for 1 h Microtubule
orientation (~
Excision at At and incubation in IAA for 1 h Microtubule
orientation (~
Lit side Shaded side Lighted side Shaded side
Unstimulated 2.3_+1.5 15_+7 20_+ 6
Controls
1 h 12.4_+2.4 84_+7 16_+10 2h 25.3_+1.8 85_+3 19_+12
85_+12 83_+ 9 13_+ 6 15_+ 8
80_+9 89• 12 12_+ 16 15 _+ 10 81_+15 13_+10 82_+ 9 17_+ 8
Table 2. Failure to induce stable micro- tubule arrays by auxin.
Maize coleoptile segments were incubated at time 0 h in water and
the resulting longitudinal microtubule array assayed for stability
against 10 pM IAA for 1 h (causing transverse orientations). For
definition of values refer to Table 1
Time after decapitation (At)
Incubation in water Microtubule orientation (0)
Incubation in water, followed by incubation in IAA Microtubule
orientation (~
Oh 18+16 19_+13 l h 86• 6 12_+16 2 h 80+21 16_+ 9 3h 78_+19 22_+
6
Table 3. Failure to induce stable microtubule arrays by
gradients of auxin in maize coleoptiles. Half of the coleoptile tip
was removed to mimick the auxin depletion produced by phototropic
stimulation. Segments were excised at variable time intervals and
the stability of
the resulting microtubule arrays (longitudinal underneath the
re- moved half of the tip and transverse underneath the remaining
half of the tip) was assayed by incubation in 10 laM IAA or water,
respectively. For definition of the values refer to Table 1
Time after decapitation (At)
Control: coleoptiles where half of the tip was removed
Induced curvature (~
Microtubule orientation (~
Excision at At and incubation in water Microtubule orientation
(~
Excision at At and incubation in IAA
Concave side Convex side Concave side Convex side Concave side
Convex side
0 h 1.9-1-2.3 10_+ 17 12_+ 16 1 h 10.7_+1.4 81_+14 19_+13 2 h
21.8 _+4.8 86_+ 13 20 _+ 19 3 h 27.9_+3.2 80_+ 15 29_+ 15
81 -+ 15 82+ 19 12-t- 16 25+ 18 79_+19 86+10 22_+ 8 16-1-14 85+_
12 83_+ 16 12+ 19 23• 18 75-t- 11 81 +21 19+ 10 16-t-22
The role of blue light and auxin in the induction of stable
microtubule arrays. It migh t be tha t the ac t ion of blue l ight
u p o n s tab i l i sa t ion of m i c r o t u b u l e a r r ays is
t rans- duced by the dep le t ion of aux in induced by the i r r ad
ia - t ion. If this were true, d e c a p i t a t i o n and
subsequent deple- t ion of e n d o g e n o u s auxin for 2 h shou
ld elicit s table mi- c ro tubu l e ar rays . One hou r after d e c
a p i t a t i o n mic ro- tubules exh ib i t ed a l ong i tud ina l
o r i en t a t i on (Table 2), bu t they read i ly r e tu rned to
the t ransverse pos i t i on after ad- d i t ion of indo le -ace t
ic acid, even as late as 3 h after de- c a p i t a t i o n (Table
2).
It was cons ide red tha t this exper iment , using symmet - ric
changes of aux in content , was only rough ly mimick - ing the
effects of un i l a te ra l b lue light. A t ransverse gradi - ent
of aux in shou ld be closer to the s i tua t ion after pho- t o t r
o p i c s t imula t ion . To p r o d u c e such a gradient , hal f
of
the coleopt i le t ip was r emoved and the o therwise in tac t
seedlings kep t up to 3 h unde r red light. F r o m 1 h after decap
i t a t ion , mic ro tubu le s were found to be long i tud ina l in
the cells sub tend ing the excised tip-half, they were t ransverse
or s l ightly obl ique in the oppos i t e co leopt i le f lank
(Table 3). However , this g rad ien t of m i c r o t u b u l e o r
i en ta t ion was erased if co leopt i le segments were incu- ba t
ed in wate r or in 0.1 m M I A A , even when this incuba- t ion was
de layed for 3 h (Table 3). Thus, all a t t emp t s to induce s
table mic ro tubu le a r r ays wi thou t blue l ight failed.
In o rde r to assess the i m p o r t a n c e of a g rad ien t of
b lue l ight for the s tab i l i sa t ion of micro tubules , an
induc- ing pulse was given symmet r i ca l ly f rom above. One or
two hours after induct ion , pu ta t ive s tabi l i ty effects were
tes ted ei ther by a un i la te ra l "coun te rpu l se" (Table 4)
or
-
P. Nick and E. Schiller: Polarity induction versus phototropism
67
Table 4. Stabilisation of microtubule orientation against
asymmet- ric blue light after symmetric irradiation of maize
coleoptiles. Plants were irradiated from above by a pulse of blue
light (1.9 gmol.m 2) and then phototropically induced by a second,
unilateral, light pulse of equal strength either 1 h (left panel)
or 2 h (right panel) after
the symmetric irradiation. Negative curvatures indicate
tropistic bending towards the second light pulse; lit and shaded
side are defined with respect to the unilateral light pulse. For
definition of orientation values refer to Table 1
Time after induction (At)
Second light pulse after 1 h
Induced curvature (~
Microtubule orientation (~
Lit side Shaded side
Second light pulse after 2 h
Induced curvature (~
Microtubule orientation (~
Lit side Shaded side
Oh + 3.3_+ 1.7 12_+ 7 18_+13 l h + 2.9_+ 0.4 79_+12 80+_23 2h
-12.8_+ 2.8 83_+23 18+_12 3 h -37.8+_ 4.9 79_+18 23_+19
12 h -98.4_+ 19.3 82_+ 14 18+_ 18
-- 0.9+_1.5 12__+19 22_+ 8 + 2.2+_0.9 76_+16 82_+18 + 0.5+1.7
85_+16 82+_15 -- 24.3 _+ 1.9 82 _+ 17 80 +_ 20 + 5.7_+4.9 79_+13
86+_22
Table 5. Stabilisation of microtubule orien- tation against
auxin after symmetric irradi- ation. Maize coleoptiles were
irradiated from above as in Table 4 and the stability of the
resulting longitudinal microtubule array questioned by incubation
in 10 gM IAA for 1 h (inducing transverse arrays). For definition
of values refer to Table 1
Time after Control: intact plants Incubation in IAA irradiation
(At) Microtubule orientation (~ Microtubule orientation (~
Oh 18_+11 10_+23 1 h 78+_16 17+_14 2h 82+_13 76_+19
by decapitation and subsequent incubation in IAA (Table 5). One
hour after vertical induction, microtubules were longitudinal in
both coleoptile fanks (Tables 4, 5). After unilateral irradiation
by the "counterpulse" they returned to the transverse array on the
side opposed to this "counterpulse" (Table 4, left). This was
accompanied by a strong curvature towards the unilateral light
pulse. A reorientation of microtubules from longitudinal to
transverse could also be achieved by incubation in IAA (Table 5,
left). Two hours after a vertical light pulse, the longitudinal
microtubule orientation had become stable against unilateral light
pulses (Table 4, right) and incuba- tions with IAA (Table 5,
right). For these conditions the unilateral "counterpulse" could
evoke only a slight, ephemeral bending response (Table 4,
right).
Induction of transverse polarity by partial illumination. If co
leopt i les were un i la te ra l ly s t imula ted over thei r ent i
re length with a pulse of 0.85 g m o l . m 2 b lue l ight ( t r ea
tment 1 in Fig. 3), a s t rong cu rva tu re of a b o u t 100 ~ t o
w a r d s the l ight pulse cou ld be obse rved 1 d la te r (control
1 in Fig. 3). A coun te rpu l se of equal s t reng th cou ld
reverse this response , if it was app l i ed up to 1 h after i nduc
t ion (curve 1 in Fig. 3). After tha t t ime the effects induced by
the first pulse h a d become s table and were expressed as a s
table cu rva tu re in the d i rec t ion of the induc ing pulse. W h
e n the same induc ing fluence was no t d i s t r ibu ted over the
ent i re length of the coleopt i le , bu t conf ined to a smal l
spo t of 0.5 m m d iame te r on the very t ip of the co leopt i le
( t r ea tment 2 and curve 2 in Fig. 3), no s tab i l i sa t ion
aga ins t the coun te rpu l se cou ld be observed, even if the coun
te rpu l se was app l i ed as late as 3 h after induct ion .
However , the p h o t o t r o p i c response, el ici ted by such a
t ip i l l umina t ion (control 2 in Fig. 3) was on ly s l ight ly
re- duced as c o m p a r e d to the response p r o d u c e d by s t
imula -
tion of the entire coleoptile (control 1 in Fig. 3). When the
inducing pulse was directed to the base of the coleoptile, 20 mm
below the tip (treatment 3 in Fig. 3), only a signif- icantly
smaller phototropic response was observed (con- trol 3 in Fig. 3).
Surprisingly, this response escaped re- versibility as early as 1 h
after induction, as fast as for irradiation of the entire
coleoptile (curve 3 in Fig. 3). Thus, with respect to induction of
transverse polarity, partial stimulation in the coleoptile base was
found to be as effective as irradiation of the entire
coleoptile.
C
Time Course of Stabilisation 200-
100-
0
.10o
.200 0
-e-,
T
i i i
1 2 3 Time Interval (h)
1 - - k tvar
__ tvr Fig. 3. Stabilization of directional memory induced by
localized stimulation. The stability of the response to the
inducing light pulse was assayed by counterdirectional stimulation
over the whole length of the coleoptile. The inducing pulse was
either distributed over the entire length of the coleoptile
(treatment 1), or in the very tip of the coleoptile (treatment 2),
or in the base, 20 mm below the tip (treatment 3). Positive
curvatures indicate bending towards the inducing pulse, negative
curvatures inversion of the response by the counterstimulus.
Controls C1 to C3 show the response for the re- spective induction
for omission of the counterpulse
-
68 P. Nick and E. Sch/ifer: Polarity induction versus
phototropism
D i s c u s s i o n
Stable arrays of microtubules and transverse polarity.
Phototropic stimulation can confer a stable transverse polarity
which controls long-term changes in growth (Nick and Schfifer 1988,
1991). It evolves from stabilizing a labile precursor, which
becomes detectable from 20 min after irradiation. This labile
precursor is based upon a gradient across the coleoptile and can be
reoriented by opposing light pulses (Nick and Sch/ifer 1991).
However, 2 h after irradiation, transverse polarity attains
resistance to counterstimulation.
A gradient across the coleoptile can be detected for the
orientation of cortical microtubules from 10 to 20 min after
phototropic stimulation, with longitudinal microtubules in the
illuminated side and transverse mi- crotubules in the shaded side
of the coleoptile (Nick et al. 1990b). It is labile and can be
reoriented by opposing light pulses (Figs. 1 and 2). However, 2 h
after irradiation, this gradient of microtubule orientation has
acquired sta- bility against opposing light pulses (Fig. 1).
Phototropic curvature and this gradient of micro- tubule
orientation are correlated with respect to time course, direction,
fluence-dependence and relation with auxin (Nick et al. 1990b,
1992). Nevertheless, they were shown to be parallel phenomena, not
causally linked to each other (Nick et al. 1991a). One important
difference is the stability of microtubule orientation beginning
from 2 h after tropistic stimulation (Nick et al. 1991a, Fig. 1).
In contrast, tropistic curvature can be transiently re- versed by
opposing gravitropic or phototropic stimuli (Nick and Sch/ifer
1988; Nick et al. 1991a,b).
On the other hand, the gradient of microtubule orien- tation and
the stable spatial memory did correlate in all cases tested so far
(Fig. 1 and Nick et al. 1991a). This includes temporal (Nick et al.
1990; Nick and Sch/ifer 1991) as well as spatial aspects. One might
argue that the stability of microtubule arrays from 2 h after
irradiation is only apparent, due to long-term sensory adaptation
or habituation (Galland 1989) inactivating the transduction chain
responsible for the blue-light action upon micro- tubule
orientation. However, the observation that micro- tubule
orientation is not only resistant to opposing blue- light pulses,
but also to changes in the content of auxin or IAA indole (Table
1), favours of a true stability of micro- tubule arrays. If
habituation were involved, it would be expected in a very late
event of transduction and should affect a step necessary for the
cellular response to auxin rather than signal transduction in sensu
stricto.
It thus appears justified to assume that microtubules are
stabilized 2 h after irradiation with blue light. The gradient of
microtubule orientation across the coleoptile might embody the
information on the direction of light- induced spatial memory. The
stability of microtubule ar- rays might be the cause for the
stability of this spatial memory. In other words: in maize
coleoptiles, light-in- duced stable arrays of microtubules might be
the cellular marker for the light-induced stable transverse
polarity.
Essential elements in the establishment of stable micro- tubule
arrays. Depletion of auxin can make microtubules
reorient in a fashion similar to blue light (Nick et al. 1990b,
1992). Thus, similar to blue light, it might endow the microtubule
arrays with stability. However, even 3 h of auxin depletion were
not able to produce stable micro- tubule arrays (Table 2). A
gradient of auxin, although able to induce a gradient in the
orientation of micro- tubules, was equally ineffective in confering
stability of this orientation (Table 3). Thus, although blue light
caus- es gradients of auxin across the coleoptile, artificially in-
duced auxin gradients could not mimick all aspects of phototropic
stimulation. Although auxin is able to trig- ger reorientation of
microtubules, stabilisation of micro- tubule arrays requires a
blue-light-induced factor, which is not auxin. A similar conclusion
has been drawn from a detailed analyses of microtubule
reorientation induced by light of different spectral qualities
(Zandomeni and Schopfer 1993). It might even be that stabilisation
of mi- crotubule arrays does not rely upon auxin at all, but
utilizes a different signal-transduction pathway. A similar
conclusion was drawn for the fixation of the physiologi- cally
defined spatial memory (Nick and Sch/ifer 1991). Thus, it appears
that blue light is essential for the stabili- sation of microtubule
arrays. The question arises whether it has to be a gradient of blue
light or whether symmetri- cal blue light has the same effect. The
answer seems to be the latter: symmetrical blue light can suppress
the re- sponses to subsequent stimuli, if its action is allowed to
develop for 2 h (Table 4). This can be seen on the physio- logical
level: only transient curvature is observed, similar to the
transient bending towards the counterpulse in Fig. 1. On the
cellular level, this becomes manifest as a stable longitudinal
microtubule array on both flanks of the coleoptile (Tables 4, 5).
This experiment directly demonstrates that the two aspects of
transverse polarity - direction and stability can be separated on
the whole- organ as well as on the cellular level. This appears to
be a general feature of polarity in plants (Jaffe 1958; Nick and
Furuya 1992). Extensive fluence-response studies on spatial memory
lead to the conclusion that the signal- transduction chains
mediating the stabilisation of trans- verse polarity and the
phototropic asymmetry leading to tropistic bending separate before
phototropic asymmetry is formed (Nick and Sch/ifer 1991). A similar
conclusion was drawn for blue-light-mediated reorientation of mi-
crotubules (Nick et al. 1992). The findings presented here suggest
that the same is true for the stabilisation of mi- crotubule arrays
(Table 5). This parallelism of the three phenomena further
strengthens the view that micro- tubule reorientation and the
stabilisation of microtubule arrays are the cellular correlates of
the physiologically defined blue-light-induced transverse
polarity.
Stable transverse polarity is a localized response. To ana- lyze
the role of longitudinal signal migration in the induC- tion of
stable transverse polarity, the stabilisation of the memory of the
direction of an inducing pulse was fol- lowed for localized
irradiation in the tip and base of the coleoptile, respectively
(Fig. 3). This experiment yielded two important results: (i) The
tropistic response to blue light can be induced best by stimulation
of the very tip of the coleoptile, as reported previously (Iino
1988). The
-
P. Nick and E. Sch/ifer: Polarity induction versus phototropism
69
tropistic response to s t imulat ion in the base of the
coleoptile is compara t ive ly weak (compare controls 1, 2 and 3 in
Fig. 3). (ii) In contrast , even as late as 3 h after induction, no
stable transverse polar i ty could be detected for unilateral tip i
r radiat ion (curve 2 in Fig. 3). However , with respect to induct
ion of stable transverse polarity, s t imulat ion at the base of
the coleoptile was as effective as s t imulat ion over the entire
coleoptile length (compare curves 1 and 3 in Fig. 3). The failure
to induce a stable polar i ty by tip i l lumination suggests that
basal cells have to see the light themselves to bring abou t this
response. In other words : no apicobasal signal can replace the di-
rect act ion of blue light. This is also valid for blue-light-
induced changes in the content of auxin. Thus, in con- trast to pho
to t rop ic curvature, stable transverse polar i ty is ce l l
-autonomous. It depends upon a signal induced by blue light, a
signal which cannot be accounted for by auxin. This is consistent
with previous experiments (Nick et al. 1990a) demons t ra t ing
that gravi t ropic s t imulat ion requires blue light to induce a
stable transverse polar i ty (Nick et al. 1990a). The spatial
separat ion of pho to t rop ic induct ion (initiated in the tip of
the coleoptile) and the induct ion of stable transverse polar i ty
(taking place in the base of the coleoptile) provides direct
evidence for the view that the two p h e n o m e n a are not
causally linked.
This work was supported by the Deutsche Forschungsgemeinschaft
and two grants of the Studienstiftung des Deutschen Volkes and the
Human Frontier Science Program Organization to P.N.
References
Galland, P. (1989) Photosensory adaptation in plants. Bot. Acta
102, 11-20
Hofmann, E., Sch/ifer, E. (1987) Red-light induced shift of the
flu- ence-response curve for first positive curvature of maize
coleop- tiles. Plant Cell Physiol. 28, 37-45
Iino, M. (1988) Pulse-induced phototropism in oat and maize
coleoptiles. Plant Physiol. 88, 823 828
Iwata, K., Hogetsu, T. (1989) Arrangement of cortical
microtubules in Arena coleoptiles and mesocotyls and Pisum
epicotyls. Plant Cell Physiol. 30, 1011-1016
Jaffe, L.F. (1958) Morphogenesis in lower plants. Annu. Rev.
Plant Physiol. 9, 359-384
Kunzelmann, P., Schfifer, E. (1985) Phytochrome-mediated pho-
totropism in maize mesocotyls. Relation between light and Pfr
gradients, light growth response and phototropism. Planta 165,
424429
Mandoli, D.F., Briggs, W.R. (1982) The photoperceptive sites and
the function of tissue light-piping in photomorphogenesis of
etiolated oat seedlings. Plant Cell Environ. 5, 137-145
Mohr, H. (1972) Lectures on photomorphogenesis. Springer,
Heidelberg Berlin New York
Nick, P., Sch/ifer, E. (1988) Spatial memory during the tropism
of maize (Zea mays L.) coleoptiles. Planta 175, 380-388
Nick, P., Sailer, H., Schfifer, E. (1990a) On the relation
between photo- and gravitropically induced spatial memory in maize
coleoptiles. Planta 181, 385-392
Nick, P., Bergfeld, R., Sch/ifer, E., Schopfer, P. (1990b)
Unilateral reorientation of microtubules at the outer epidermal
wall during photo- and gravitropic curvature of maize coleoptiles
and sun- flower hypocotyls. Planta 181, 162-168
Nick, P., Schfifer, E. (1991) Induction of transverse polarity
by blue light: An all-or-none response. Planta 185, 415424
Nick, P., Furuya, M., Sch/ifer, E. (1991a) Do microtubules
control growth in tropism? Experiments with maize coleoptiles.
Plant Cell Physiol. 32, 999-1006
Nick, P., Sch/ifer, E., Hertel, R., Furuya, M. (1991b) On the
putative role of microtubules in gravitropism of maize coleoptiles.
Plant Cell Physiol. 32, 873-880
Nick, P., Furuya, M. (1992) Induction and fixation of polarity
Early steps in plant morphogenesis. Develop. Growth Differ. 34,
115-125
Nick, P., Sch/ifer, E., Furuya, M. (1992) Auxin redistribution
during first positive phototropism in corn coleoptiles. Microtubule
re- orientation and the Cholodny-Went theory. Plant Physiol. 99,
130~1308
Robinson, D.G., Quader, H. (1982) The microtubule-microfibril
syndrome. In: The cytoskeleton in plant growth and develop- ment,
pp. 109-126, Lloyd, C.Wo, ed. Academic Press, London
Sakiyama, M., Shibaoka, H. (1990) Effects of abscisic acid on
the orientation and cold stability of cortical microtubules in epi-
cotyl cells of the dwarf pea. Protoplasma 157, 165-171
Zandomeni, K., Schopfer, P. (1993) Reorientation of microtubules
at the outer epidermal wall of maize coleoptiles by phy- tochrome,
blue-light receptor and gravity. Protoplasma 173, 103-112